Eph Signaling in Vascular Development Thesis by Sebastian de la Soudiere Gerety In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology California Institute of Technology Pasadena, CA 2002 (Submitted October 5th, 2(01) 11 Acknowledgements 1 would like to thank David for providing an intellectually rigorous environment in which to learn both the skills and patience necessary for success in research. In the last 6 years, we have seen him grow as a mentor to his lab, and as a person, as the people and circumstances around him have changed. I wish David the best of luck with his new scientific directions into the study of pain, and fear. I would also like to thank Scott Fraser for our regular morale boosting conversations, for his passion for neat science, and the calm he can engender in anxious graduate students. Thanks also to Marianne Bronner-Fraser, for her honesty, openness, and for helping me find my way out of here! Thanks to the rest of my committee, Kai, and Paul, for accepting my bribes of doughnuts and fruit that got me through candidacy. I would like to thank the Caltech biology department for their flexibility, which helps make our 6,7, or 10 years of slave-labor easier to bear. A major source of inspiration has of course been my labmates, who are always there. 24-7. Thanks to my fellow angiogenesis subgroupers, who put up with twice-monthly ranting about my supposed lack of data, and gave me useful ideas for my work, even if they didn't always know they were. I want to thank Nirao for sharing his passion for the crest, and inspiring me to be rigorous, and rude. Thanks also to Andy, for passively encouraging me to do things even though they hurt, with a smile. Thanks to Sherry and Amy, who tried to tell me life was worth living, in their own ways. Thanks to LiChing for always standing up for herself and others. Qiufu Ma's innocent kindness has been sorely missed in the lab for many years, and Jae's chronic cynicism will stay with me for a long time. Thanks to the tech staff, especially Joanna, who always had lots of smiles to share with to all of us. Thanks to Shirley and Bruce, for their generosity, and for dealing with mouse politics with a smile. Thanks to Gina and Gaby for trying to smile while doing their jobs. Thanks to Mariela for being a good friend, best of luck to you and Tobi, III who will no doubt share lots of love together. Thanks to my family, for blindly worshipping science and the people who do it, and their respect for my priorities in life. Mom and Pop have always been an inspiration. The biggest thanks of all goes to Emma, without whom I would probably not have made it. She came at the worst time in my life, and tloated me back to the surface with love and compassion. It lasts to this day. I can't wait to get to Oxford! IV ABSTRACT One of the most striking features of developmental biology is the dramatic morphological changes that an embryo must undergo to achieve its final form. Arguably, the most stunning example of this is found in the embryonic vasculature: not only does the vasculature undergo morphological changes, it must continue to do so adaptively as the size and nutritional needs of the embryo change during gestation. As embryonic blood flow starts long before the end of vessel morphogenesis, the vessels must maintain the integrity of their cell-cell contacts while at the same time remodeling into their final state. Receptor tyrosine kinases and their ligands have been implicated in the rcgulation of blood vessel growth and remodeling during development. Recently, the Eph receptors and their ephrin ligands were found to be expressed in the developing vasculature. While one Eph receptor, EphB4, is restricted to veins, its specific ligand, ephrinB2, is restricted to arteries. Furthermore, the ephrinB2 knockout mice exhibit defects in blood vessel remodeling, angiogenesis. Although the reciprocal expression of ephrinB2 and EphB4 suggested that Eph signaling from arteries to veins was important for blood vessel development, the presence of additional Eph receptors suggested EphB4 might not be required for this process. Additionally, the widespread expression of ephrinB2 outside the vasculature suggested that vascular-specific expression of this ligand might not be the tissue source necessary for angiogenic remodeling. To determine which Eph receptor was mediating the ephrinB2 signal, I generated a knockout of the EphB4 gene in mouse. A reporter gene replacement in the EphB4 locus confirmed the vein-biased expression of this receptor. Homozygous EphB4 mutant mice exhibit angiogenesis and cardiac defects, and embryonic lethality indistinguishable from those of ephrinB2 knockout mice. This suggests that EphB4 is the main Eph receptor responsible for transducing the angiogenic ephrinB2 signal. To examine the importance of endothelial specific expression of ephrinB2 in angiogenesis, in contrast to its non­ vascular expression, I generated a conditional ephrinB2 mouse. These mice carry a functional ephrinB2 gene, which can be inactivated in a tissue specific manner. Mice with endothelial-specific inactivation of ephrinB2 (and intact non-vascular ephrinB2 expression) exhibit severe angiogenesis and cardiac defects identical to those of the v conventional ephrinB2 mutant mice. This suggests that vascular ephrinB2 is essential, and cannot be compensated for by non-vascular ephrinB2 from surrounding vessels. These studies have clarified two important issues. The first is that the ephrinB2 signal is received by EphB4 expressing endothelial cells (of the veins), rather than by perivascular cells that also express Eph receptors. Second, cphrinB2 expression in endothelial cells of the vessels is an essential tissue source of angiogenic ephrin signals. Together, these studies reinforce the original interpretation of the ephrinB2 mutant, that Eph signaling between arteries to veins is essential for angiogenesis in the early embryo. VI TABLE OF CONTENTS Acknowledgements .. ...................................................................................................... .ii Abstract. ....................................................................................................................... .. .i V Table of Contents ........................................................................................................... vi Chapter 1 Introduction ............................................................................................................... 1 References ................................................................................................................ 33 Chapter 2 Symmetrical Mutant Phenotypes of the Receptor EphB4 and Its Specific .............. 51 Transmembrane Ligand ephrin-B2 in Cardiovascular Development Chapter 3 Cardiovascular EphrinB2 Function is Essential for Embryonic Angiogenesis ....... 66 References ....................................... '" ..................................................................... 90 Chapter 4 Concluding Remarks .............................................................................................. 115 References .............................................................................................................. 123 Appendix I Preliminary Comparative Analysis of EphB4 and EphrinB2 Expression ............. 129 References ............................................................................................................. 138 Appendix II Expression of Ephrin-B2 Identifies a Stable Genetic Difference Between Arterial and Venous Vascular Smooth Muscle as Well as Endothelial Cells, and Marks Subsets of Microvessels at Sites of Adult Neovascularization ... 142 1 Chapter 1 Introduction 2 Angiogenesis, the development of the vasculature, IS an essential ongolllg event during embryonic development. Simple diffusion of nutrients and waste becomes insufficient as the size of the embryo grows and it therefore becomes dependent on the vasculature to provide rapid, distributed supply of nutrients, oxygen, and waste removal. As the embryo grows, so must the vasculature, adaptively as thc needs of the embryo change, and all while maintaining luminal integrity to ensure proper confinement and transport of blood. The endothelial cells (ECs) that form the inner layer of all vessels require careful coordination of their morphological, proliferative, migratory, and adhesive activities to guarantee proper growth of the embryo and establishment of a functional mature vascular network (Risau, 1997). This is mediated by a variety of signaling pathways involving secreted and cell-surface ligands and receptors. Figuring prominently is the presence of endothelial specific receptor tyrosine kinases (RTKs) (Gale and Yancopoulos, 1999). Recent progress in the field of angiogenesis has revealed the importance of the Eph family of RTKs and their cell-surface tethered ligands, the ephrins (Adams and Klein, 2000; Yancopoulos et aI., 2000; Gale and Yancopoulos, 1999). Known primarily for their roles in tissue domain segregation and repulsive activities on migratory cells and pathfinding axons (reviewed in Wilkinson, 2000), they are also expressed in and around the endothelium of the vasculature (Adams et aI., 1999; Wang et aI., 1998). Initial mouse knockout studies have indicated that Eph signaling is essential for angiogenesis (Adams and Klein, 2000; Adams et aI., 1999; Wang et aI., 1998). This family of RTKs, EphB4 (Andres et aI., 1994; Bennett et aI., 1994) and its specific ligand ephrinB2